Copper release by MOF-74(Cu): a novel pharmacological alternative to diseases with deficiency of a vital oligoelement

Copper deficiency can trigger various diseases such as Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease (PD) and even compromise the development of living beings, as manifested in Menkes disease (MS). Thus, the regulated administration (controlled release) of copper represents an alternative to reduce neuronal deterioration and prevent disease progression. Therefore, we present, to the best of our knowledge, the first experimental in vitro investigation for the kinetics of copper release from MOF-74(Cu) and its distribution in vivo after oral administration in male Wistar rats. Taking advantage of the abundance and high periodicity of copper within the crystalline-nanostructured metal–organic framework material (MOF-74(Cu)), it was possible to control the release of copper due to the partial degradation of the material. Thus, we simultaneously corroborated a low accumulation of copper in the liver (the main detoxification organ) and a slight increase of copper in the brain (striatum and midbrain), demonstrating that MOF-74(Cu) is a promising pharmacological alternative (controlled copper source) to these diseases.


Characterization methods
X-ray diffraction (XRD) patterns were obtained using a D8 Advance (Bruker®), with a copper Kα radiation source.Thermal stability of samples was followed by Thermogravimetric Analysis (TGA) in equipment by TA Instruments®.The determination of particle size for MOF-74(Cu), 1.8 microns, was determined by dispersing a sample of the Cu(II)-MOF material in methanol and analyzed by dynamic light scattering (DLS) (Litesizer 500 Anton Paar®).

Release profiles
For in-vitro experiments, a known quantity of MOF-74(Cu) was immersed in 8 mL of preheated phosphate-buffered saline (PBS) solution (0.1M) at pH 1,2 for 10 minutes, to simulate the gastric emptying of the rat and subsequently changed to a solution of pH 7.4 similar to the conditions of the intestine.Vials were sealed and maintained at 37°C±1°C and stirred.An aliquot of 100 µL was withdrawn at different times (10, 20, 30, 60, 120, 240, 300,  and 360 min) and replaced with the same volume of fresh dissolution medium.The aliquots were filtered by a 0.2 µm pore size membrane.

Animals
Male Wistar rats weighing between 260 and 290 g (8.8 ± 0.35 weeks old) were used, and these were provided by the Animals Laboratory Production and Experimentation Unit (UPEAL for its Spanish acronym) from the Metropolitan Autonomous University-Xochimilco (UAM-X, Mexico).The animals were housed under standard laboratory conditions with a 12 h light/dark cycle and had ad-libitum access to food (Laboratory Rodent Diet 501) and water.All experimental procedures were approved by the Internal Committee for the Care and Use of Laboratory Animals from the same institution (Protocol No. 170).Additionally, the guidelines of the Official Mexican Standard (NOM-062-ZOO-1999) from the Secretariat of Agriculture, Livestock Rural Development, Fisheries and Food (1999) were followed.All tests were performed during daylight hours, and the number of experimental animals was kept at a minimum (n=3-4).

Oral administration of MOF-74(Cu)
A dispersion of MOF-74(Cu) equivalent to doses of 39.75(D1) or 79.5 mg/kg (D2) of Cu(II) was administered by oral gavage to different groups of rats.After that, the groups that received D1 were sacrificed by decapitation at 3, 6, 9, and 12 h post-treatment.While the groups treated with the higher dose (D2) were sacrificed at 6 and 12 h.Samples of the blood, striatum, midbrain, liver (left lateral lobe), kidney, and spleen were collected at each time.Plasma samples were obtained by blood centrifugation at 3500 rpm for 10 min and stored at − 21 °C until copper determination.The brain tissues were dissected on ice and immediately weighed.Animals in the control group received the vehicle (deionised water) and equimolar doses of H2BDC-OH, (2,5-dioxide terephthalate, DOT), which were sacrificed immediately (zero time).The amount of copper found in the different tissues and blood samples of the control group was considered the baseline value.All samples were kept at -21 °C until analysis.

Copper quantification assay
Copper (I and II) determination was performed using an atomic absorption spectrophotometer (AA) (Perkin-Elmer® 3110) at a wavelength of 324.8 nm and equipped with an HGA-600 graphite furnace and an AS-60 autosampler.Copper concentrations were calculated using a six-point calibration curve over a copper concentration range of 2 to 45 µg/L copper, using the standard dilution method with 0.2% Nitric acid 65% Suprapur®.All the curves presented a coefficient of determination ≥ 0.99.The method was validated to demonstrate whether it complies with the identification and quantification of the metal, in a precise, exact, and robust manner.Tissue samples: acid digestion of the different tissues was performed by adding 0.3 mL of supra pure HNO3 and shaking the samples in a water bath at 60 °C for 30 min.The total charge of Cu(II) in the MOF-74(Cu): 2 mg of MOF-74(Cu) were weighed in triplicate, and 5 mL of a 1M H3PO4 solution was added.The resultant mix was shacked in a water bath for 24 h at 50°C and 60 rpm.All samples were diluted in 0.2% v/v with supra pure HNO3.Results were reported in micrograms of copper per gram of wet tissue (µg/g) for tissues and micrograms per milliliter (µg/mL) for plasma samples and the samples obtained from release profiles tests.

Statistical analysis
One-way analysis of variance (ANOVA) followed by a Dunnett´s test was used for data with normal distribution and variance homogeneity.For data that did not meet the requirements for parametric analysis results Kruskal-Wallis and Mann-Whitney-U tests were used, establishing statistical significance at *p<0.05 for all tests.

MOF-74(Cu) characterization
Figure S1 shows the PXRD pattern of MOF-74(Cu).PXRD patterns display crystalline structures fitting to that of MOF-74(M) previously reported. [1]The TGA profile shown in Figure S2 confirms the low thermal stability of MOF-74(Cu).The sample presents a weight loss close to 23%, in the temperature range between 30 and 100°C, which is expected.In the following temperature range, the MOF-74(Cu) sample shows an additional loss of close to 5%.So that it finally degrades with greater weight loss from 234°C.In comparison, the mean particle size is shown in Table S1.

*
The data shown is the result of 5 measurements for each sample.